DB code: S00315

RLCP classification 3.113.163000.1110 : Transfer
3.1143.70030.3060 : Transfer
CATH domain 3.40.50.620 : Rossmann fold Catalytic domain
E.C. 6.3.1.5
CSA
M-CSA
MACiE M0200

CATH domain Related DB codes (homologues)
3.40.50.620 : Rossmann fold S00314 S00549 S00316 S00317 S00318 T00085 T00249 D00300 M00177 M00178 T00106 T00114

Uniprot Enzyme Name
UniprotKB Protein name Synonyms RefSeq Pfam
P08164 NH(3)-dependent NAD(+) synthetase
EC 6.3.1.5
Spore outgrowth factor B
Sporulation protein outB
General stress protein 38
GSP38
NP_388195.1 (Protein)
NC_000964.3 (DNA/RNA sequence)
PF02540 (NAD_synthase)
[Graphical View]

KEGG enzyme name
NAD+ synthase
NAD+ synthetase
NAD+ synthase
nicotinamide adenine dinucleotide synthetase
diphosphopyridine nucleotide synthetase

UniprotKB: Accession Number Entry name Activity Subunit Subcellular location Cofactor
P08164 NADE_BACSU ATP + deamido-NAD(+) + NH(3) = AMP + diphosphate + NAD(+). Homodimer.

KEGG Pathways
Map code Pathways E.C.
MAP00760 Nicotinate and nicotinamide metabolism
MAP00910 Nitrogen metabolism

Compound table
Cofactors Substrates Products Intermediates
KEGG-id C00305 C00238 C00002 C00857 C00014 C00020 C00013 C00003
E.C.
Compound Magnesium Potassium ATP Deamido-NAD+ NH3 AMP Pyrophosphate NAD+ NAD-adenylate
Type divalent metal (Ca2+, Mg2+) univalent metal (Na+, K+) amine group,nucleotide amine group,carbohydrate,nucleotide amine group,organic ion amine group,nucleotide phosphate group/phosphate ion amide group,amine group,nucleotide
ChEBI 18420
 18420
 		29103
 29103
 		15422
 15422
 		18304
 18304
 		16134
 16134
 		16027
 16027
 		29888
 29888
 		15846
 15846
PubChem 888
 888
 		813
 813
 		5957
 5957
 		165491
 165491
 		222
 222
 		6083
 6083
 		1023
 21961011
 1023
 21961011
 		5893
 5893
1ee1A Bound:_MG Unbound Bound:ATP Bound:DND Unbound Unbound Unbound Unbound Unbound
1ee1B Unbound Unbound Unbound Bound:DND Unbound Unbound Unbound Unbound Unbound
1fydA Bound:_MG Unbound Unbound Unbound Unbound Bound:AMP Bound:POP Unbound Unbound
1fydB Unbound Unbound Unbound Unbound Unbound Unbound Unbound Unbound Unbound
1ifxA Unbound Unbound Unbound Bound:DND Unbound Unbound Unbound Unbound Unbound
1ifxB Unbound Unbound Unbound Bound:DND Unbound Unbound Unbound Unbound Unbound
1ih8A Bound:2x_MG Unbound Analogue:APC Unbound Unbound Unbound Unbound Unbound Unbound
1ih8B Bound:_MG Unbound Unbound Unbound Unbound Unbound Unbound Unbound Unbound
1kqpA Bound:2x_MG Unbound Unbound Unbound Unbound Unbound Bound:POP Unbound Intermediate-bound:ADJ
1kqpB Bound:2x_MG Unbound Unbound Unbound Unbound Unbound Bound:POP Unbound Intermediate-bound:ADJ
1nsyA Bound:_MG Unbound Unbound Unbound Unbound Bound:AMP Bound:POP Analogue:ATP Unbound
1nsyB Bound:_MG Unbound Unbound Unbound Unbound Bound:AMP Bound:POP Analogue:ATP Unbound
2nsyA Bound:2x_MG Unbound Unbound Unbound Unbound Unbound Bound:POP Unbound Intermediate-bound:__A-NAD
2nsyB Bound:2x_MG Unbound Unbound Unbound Unbound Unbound Bound:POP Unbound Intermediate-bound:__A-NAD

Reference for Active-site residues
resource references E.C.
Swiss-prot;P08164 & literature [4], [6], [7]

Active-site residues
PDB Catalytic residues Cofactor-binding residues Modified residues Main-chain involved in catalysis Comment
1ee1A SER 46;ASP 173 ASP 50;GLU 162(Magnesium-1 binding);THR 208(Magnesium-2);ASP 220(Potassium)
1ee1B SER 46;ASP 173 ASP 50;GLU 162(Magnesium-1 binding); ; invisible 205-225
1fydA SER 46;ASP 173 ASP 50;GLU 162(Magnesium-1 binding);THR 208(Magnesium-2);ASP 220(Potassium)
1fydB SER 1046;ASP 1173 ASP 1050;GLU 1162(Magnesium-1 binding); ; invisible 1205-1225
1ifxA SER 46;ASP 173 ASP 50;GLU 162(Magnesium-1 binding); ; invisible 205-225
1ifxB SER 1046;ASP 1173 ASP 1050;GLU 1162(Magnesium-1 binding); ; invisible 1205-1225
1ih8A SER 46;ASP 173 ASP 50;GLU 162(Magnesium-1 binding);THR 208(Magnesium-2);ASP 220(Potassium)
1ih8B SER 1046;ASP 1173 ASP 1050;GLU 1162(Magnesium-1 binding); ; invisible 1205-1224
1kqpA SER 46;ASP 173 ASP 50;GLU 162(Magnesium-1 binding);THR 208(Magnesium-2);ASP 220(Potassium)
1kqpB SER 46;ASP 173 ASP 50;GLU 162(Magnesium-1 binding);THR 208(Magnesium-2);ASP 220(Potassium)
1nsyA SER 46;ASP 173 ASP 50;GLU 162(Magnesium-1 binding);THR 208(Magnesium-2);ASP 220(Potassium)
1nsyB SER 46;ASP 173 ASP 50;GLU 162(Magnesium-1 binding);THR 208(Magnesium-2);ASP 220(Potassium)
2nsyA SER 46;ASP 173 ASP 50;GLU 162(Magnesium-1 binding);THR 208(Magnesium-2);ASP 220(Potassium)
2nsyB SER 46;ASP 173 ASP 50;GLU 162(Magnesium-1 binding);THR 208(Magnesium-2);ASP 220(Potassium)

References for Catalytic Mechanism
References Sections No. of steps in catalysis
[1]
p.5130-5131
[4]
Fig.7, p.1134-1138
[7]
p.1143-1145, Fig.6

References
[1]
Resource
Comments X-RAY CRYSTALLOGRAPHY (2.0 ANGSTROMS).
Medline ID 97050817
PubMed ID 8895556
Journal EMBO J
Year 1996
Volume 15
Pages 5125-34
Authors Rizzi M, Nessi C, Mattevi A, Coda A, Bolognesi M, Galizzi A
Title Crystal structure of NH3-dependent NAD+ synthetase from Bacillus subtilis.
Related PDB 1nsy
Related UniProtKB P08164
[2]
Resource
Comments
Medline ID
PubMed ID 8916230
Journal Proteins
Year 1996
Volume 26
Pages 236-8
Authors Rizzi M, Nessi C, Bolognesi M, Coda A, Galizzi A
Title Crystallization of NAD+ synthetase from Bacillus subtilis.
Related PDB
Related UniProtKB
[3]
Resource
Comments
Medline ID
PubMed ID 9261082
Journal Structure
Year 1997
Volume 5
Pages 895-906
Authors Savage H, Montoya G, Svensson C, Schwenn JD, Sinning I
Title Crystal structure of phosphoadenylyl sulphate (PAPS) reductase: a new family of adenine nucleotide alpha hydrolases.
Related PDB
Related UniProtKB
[4]
Resource
Comments X-RAY CRYSTALLOGRAPHY (2.0 ANGSTROMS).
Medline ID 98428669
PubMed ID 9753692
Journal Structure
Year 1998
Volume 6
Pages 1129-40
Authors Rizzi M, Bolognesi M, Coda A
Title A novel deamido-NAD+-binding site revealed by the trapped NAD-adenylate intermediate in the NAD+ synthetase structure.
Related PDB 2nsy
Related UniProtKB P08164
[5]
Resource
Comments
Medline ID
PubMed ID 10544053
Journal J Struct Biol
Year 1999
Volume 127
Pages 279-82
Authors Ozment C, Barchue J, DeLucas LJ, Chattopadhyay D
Title Structural study of Escherichia coli NAD synthetase: overexpression, purification, crystallization, and preliminary crystallographic analysis.
Related PDB
Related UniProtKB
[6]
Resource
Comments X-ray crystallography
Medline ID
PubMed ID 11375500
Journal Acta Crystallogr D Biol Crystallogr
Year 2001
Volume 57
Pages 806-12
Authors Devedjiev Y, Symersky J, Singh R, Jedrzejas M, Brouillette C, Brouillette W, Muccio D, Chattopadhyay D, DeLucas L
Title Stabilization of active-site loops in NH3-dependent NAD+ synthetase from Bacillus subtilis.
Related PDB 1ee1 1fyd 1ifx 1ih8
Related UniProtKB
[7]
Resource
Comments X-ray crystallography
Medline ID
PubMed ID 12077433
Journal Acta Crystallogr D Biol Crystallogr
Year 2002
Volume 58
Pages 1138-46
Authors Symersky J, Devedjiev Y, Moore K, Brouillette C, DeLucas L
Title NH3-dependent NAD+ synthetase from Bacillus subtilis at 1 A resolution.
Related PDB 1kqp
Related UniProtKB

Comments
This enzyme catalyzes two successive transfer reactions as follows;
The first reaction: Transfer of adenylate (AMP) to carboxylate of deamido-NAD, releasing pyrophosphate.
The second reaction: Transfer of acyl group of NAD to amine of ammonia molecule, releasing AMP and H2O.
According to the literature [1] & [4], the first reaction activates the substrate, thorough the adenylation. The literature [4] proposed the catalytic mechanisms for these two transfer reactions.
The first transfer reaction, in which no protein residues are directly involved in the catalysis, proceeds as follows:
(1) Two Mg2+ ions stabilize the leaving pyrophosphate, by neutralizing the negative charges, and also activate the transferred group, alpha-phosphate of ATP, by enhancing the electrophilicity of the group through polarization.
(2) The acceptor group, the oxygen atom of the carboxylate attached to nicotin, makes a nucleophilic attack on the transferred group, the alpha-phosphate of ATP.
(3) The two Mg2+ ions and a monovalent cation (K+) stabilize the pentacovalent transition-state.
(4) Adenylated-NAD intermediate is formed, releasing the pyrophosphate.
The second reaction proceeds as follows:
(1') Asp173 acts as a general base, to deprotonate the ammonium ion.
(2') The activated ammonium makes a nucleophilic attack on the carbonyl group of the adenylated-NAD intermediate, forming a tetrahedral transition-state adduct.
(3') The protonated sidechain of Asp173 and the monovalent cation (K+) stabilize the tetrahedral transition-state.
(4') Finally, NAD and AMP are formed.
According to the literature [7], the carbonyl group of the adenylated-NAD might be protonated due to an electron-withdrawing effect by the monovalent cation, such as K+ ion.

Created Updated
2004-04-19 2009-02-26